Preparative scale conversion of gonyautoxins to neosaxitoxin

ABSTRACT

A method of preparing neosaxitoxin in quantities of a purity sufficient to allow the compound to be used as an active pharmaceutical ingredient (API) is described. The method includes the reductive desulfonation of an unresolved mixture of gonyautoxin 1 (GTX1) and gonyautoxin 4 (GTX4).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International ApplicationNo. PCT/IB2019/058019, filed on Sep. 23, 2019, which claims priority ofInternational Application No. PCT/IB2018/057274, filed on Sep. 21, 2018.The contents of each of these applications are incorporated by referencein their entirety.

TECHNICAL FIELD

The invention relates to the conversion of a mixture of gonyautoxin 1(GTX1) and gonyautoxin 4 (GTX4) to neosaxitoxin (neoSTX) on apreparative scale and the purification of the neoSTX for use as anactive pharmaceutical ingredient (API).

BACKGROUND ART

As stated in the publication of Garcia-Altares (2017), marine microalgaltoxins constitute one of the most diverse and sophisticated groups ofnatural products. Examples are paralytic shellfish toxins (PSTs) such assaxitoxin (STX), its analogues and derivatives. Gonyautoxins (GTXs) aresulphated analogs of STX and marine bacteria can transform GTXs into STXthrough reductive eliminations. In marine environments the mainproducers of STX are eukaryotic dinoflagellates.

STX is a monoterpenoid indole alkaloid containing a tricyclic3,4-propinoperhydropurine system with 2 guanidinium moieties formed bythe NH₂ groups in the positions C2 and C8 of the reduced purine:

STX blocks voltage-gated sodium channels (VGSCs), but also binds tocalcium and potassium channels. The nature of the substituents greatlyinfluences the overall toxicity of saxitoxin analogues. Thehydroxylation of Ni, e.g. as in neosaxitoxin (neoSTX), does not play amajor role in binding affinity, but seems to increase potency.

The prior art is replete with disclosures of the cosmetic andtherapeutic applications of PSTs, including their use as localanesthetics and analgesics. The publication of Mezher (2018) disclosesthat the US Food and Drug Administration (FDA) plans to develop guidancedocuments to encourage the development of extended-release localanesthetics which could replace the need for systemic oral opioids incertain situations. The expectations of the US FDA are for thedevelopment of new non-opioid drugs to treat chronic pain that couldprovide a safer alternative for patients who require long-term use ofanalgesic drugs. The publications of Kohane et al (2000),Rogriguez-Navarro et al (2011), Templin et al (2015) and Wylie et al(2012) disclose the use of neoSTX in these applications. A limitation onthe exploitation and widespread adoption of these applications is theavailability of the PSTs in sufficient quantity and of sufficient purityto render their use in the manufacture of pharmaceutical preparationscommercially viable.

The following publications include disclosures of the preparation ofgonyautoxin 1 (GTX1), gonyautoxin 4 (GTX4) or neosaxitoxin (neoSTX).Often the preparation is on an analytical scale, or does not provide thequantity and purity required for use of the preparation as an activepharmaceutical ingredient (API).

The publication of Hall et al (1984) discloses the confirmation by x-raycrystallography of the position and identity of the three substituentswhich, with the parent compound, form the array of twelve saxitoxinsfound in protogonyaulax.

The publication of Daigo et al (1985) discloses the extraction andisolation of neosaxitoxin (neoSTX) from specimens of crab. Thedose-death time curve obtained for the isolated neoSTX was clearlydistinguishable from the curve for saxitoxin (STX).

The publication of Laycock et al (1994) discloses methods for thepurification of some of the common paralytic shellfish poisoning (PSP)toxins in quantities sufficient for use as analytical standards. The PSPtoxins were purified from the dinoflagellate Alexandrium excavatum, thegiant sea scallop (Plagopecten magellanicus) hepatopancreas and thecyanobacterium Aphanizomenon flos-aquae.

The publication of Ravn et al (1995) discloses what are asserted to beoptimal conditions for extraction of paralytic shellfish toxins from aclone of Alexandrium tamarense. The paralytic shellfish toxins areextracted with acetic acid and hydrochloric acid in the concentrationrange 0.01 to 1.0 N.

Concentrations of hydrochloric acid in the range 0.03 to 1.0 N wereobserved to cause the amount of C1 and C2 toxins to decrease sharplywith a concomitant increase in the amount of gonyautoxins 2 (GTX2) and 3(GTX3).

The publication of Tsai et al (1997) discloses the detection ofparalytic toxicity by a tetrodotoxin bioassay in specimens of crab.Partial purification and characterisation of the toxins demonstrated themain toxin to be tetrodotoxin with minor amounts of gonyautoxins (GTXs)and neosaxitoxin (neoSTX).

The publication of Siu et al (1997) discloses the examination of theeffects of environmental and nutritional factors on population dynamicsand toxin production in Alexandrium catenella. Optimum conditions forthe growth of this species of dinoflagellate are disclosed along withthe toxin profile for a species grown under these conditions. The toxinprofile as detected by HPLC was found to include in descending orderGTX4, GTX3, GTX1, B2, neosaxitoxin (neoSTX) and saxitoxin (STX).

The publication of Sato et al (2000) discloses the transformation of theO-sulfate group of GTX1 and GTX4 to methylene to form neosaxitoxin. Thetransformation was achieved using thiols such as glutathione andintermediates of the conversion were isolated.

The publication of Parker et al (2002) discloses an investigation of theautotrophic growth of the toxic dinoflagellate, Alexandrium minutum, inthree different high biomass culture systems, assessing growth,productivity and toxin production. The organism was grown in aerated andnon-aerated two litre Erlenmeyer flasks, 0.5 litre glass aerated tubes,and a four litre lab scale alveolar panel photobioreactor. A markedincrease in biomass and productivity in response to aeration wasobserved. A maximum cell concentration of 3.3×10⁵ cells/mL, a meanproductivity of 0.4×10⁴ cells/mL/day and toxin production ofapproximately 20 μg/L/day with weekly harvesting was reported.

The publication of Baker et al (2003) discloses the production bybacterial strains isolated from saxitoxin-producing dinoflagellates ofcompounds that could easily be mistaken for gonyautoxin 4 (GTX4).

The publication of Miao et al (2004) discloses the isolation ofgonyautoxins (GTX1, GTX2, GTX3 and GTX4) from two strains of Alexandriumminutum Halim. The strain of Alexandrium minutum Halim designated Amtk4is asserted to be suitable for the preparation of gonyautoxins.

The publication of Jiang and Jiang (2008) discloses the establishment ofoptimal conditions for the extraction of paralytic shellfish poisoningtoxins from the gonad of Chlamys nobilis. The extraction uses aceticacid and hydrochloric acid in the concentration range of 0.04 to 1.0mol/L. The use of hydrochloric acid in the concentration range of 0.25to 1.0 mol/L was shown to cause a significant decrease of the toxins C1,C2 and GTX5 and the concomitant increase in the toxins GTX2,3. Theamount of the three unstable toxins did not show any change when aceticacid was used in the extraction.

The publication of Liu et al (2010) discloses the culture of toxinproducing Alexandrium catenella in the laboratory. A maximum celldensity of 0.4×10⁴ cells/mL was obtained within eight days of culture.Analysis by high performance liquid chromatograph (HPLC) of a crudeextraction showed the major toxic components to be C1/2, GTX4, GTX5 andneoSTX at concentrations of about 0.04550, 0.2526, 0.3392, 0.8275 and0.1266 μmol/L, respectively.

The publication of Foss et al (2012) discloses a comparison ofextraction methods for paralytic shellfish toxins (PSTs) from thefilamentous cyanobacterium Lyngbya wollei. In the absence ofcommercially available standards for the unique toxins produced by thiscyanobacterium it was not possibly to quantify the toxins extracted.

The publication of Li et al (2013) discloses a method for the rapidscreening and identification of paralytic shellfish poisoning (PSP)toxins in red tide algae. The method utilises hydrophilic interactionchromatography-high resolution mass spectrometry (HILIC-HR-MS) combinedwith an accurate mass database. Limits of detection (LOD) of ten commonPSP compounds were in the range of 10 to 80 nmol/L. The developed methodwas asserted to be a useful tool for the rapid screening and qualitativeidentification of common PSP toxins in harmful algae.

The publication of Bernardi Bif et al (2013) discloses the sensitivityof sea urchins to toxic cell extracts containing saxitoxins.

The publication of Poyer et al (2015) discloses the development of ananalytical method to characterise and differentiate saxitoxin analogs,including sulfated (gonyautoxins) and non-sulfated analogs. Hydrophilicinteraction liquid chromatography (HILIC) was used to separate sulfatedanalogs. Ion mobility mass spectrometry (IM-MS) was used as a newdimension of separation based on ion gas phase confirmation todifferentiate the saxitoxin analogs. Positive and negative ionisationmodes were used for gonyautoxins. Positive ionisation mode was used fornon-sulfated analogs. The coupling of three complementary techniques,HILIC-IM-MS, permitted the separation and identification of saxitoxinanalogs, isomer differentiation being achieved in the HILIC dimensionwith non-sulfated analogs separated in the IM-MS dimension.

The publication of Rubio et al (2015) discloses a method to purifysaxitoxin using a liquid chromatography methodology based on ionicpairs. The saxitoxin is extracted using hydrochloric acid and treatedwith ammonium sulfate following a treatment with trichloroacetic acidand hexane/diethyl ether (97/3). Samples were analysed by asemi-preparative HPLC in order to collect pure fractions of saxitoxinand these fractions were eluted in solid-phase cationic interchange STXextraction columns. The purified saxitoxin was reported to be stable andhomogenous and its identity confirmed by LC-MS-MS. Analogs such asneosaxitoxin of a decarbamoyl saxitoxin were reported to be absent fromthe purified saxitoxin.

The publication of Chen et al (2016) discloses the application of serialcoupling of reverse-phase liquid chromatography (RPLC) and hydrophilicinteraction chromatography (HILIC) combined with high resolution massspectrometry (HR-MS) to the simultaneous screening and identification ofknown lipophilic and hydrophilic toxins in the algae of harmful algalblooms (HABs). Lipophilic and hydrophilic toxins were extractedsimultaneously by the use of ultrasound-assisted extraction (UAE). Thepublication demonstrated that HPLC/HILIC-HR-MS combined with an accuratemass list of known marine algal toxins may be used as a powerful toolfor screening of different classes of known toxins in marine harmfulalgae.

The publication of Cho et al (2016) discloses the analysis of crudeextracts of toxin-producing dinoflagellates by column switching andtwo-step gradient elusion using hydrophilic-interaction chromatograph(HILIC) combined with mass spectrometry. The publication states that thedata obtained supports the hypothesis that the early stages of thesaxitoxin biosynthesis and shunt pathways are the same indinoflagellates and cyanobacteria.

The publication of Beach et al (2018) discloses the sensitive multiclassanalysis of paralytic shellfish toxins, tetrodotoxins and domoic acid inseafood using a capillary electrophoresis (CE)-tandem mass spectrometry(MS/MS) method. A novel, highly acidic background electrolyte comprising5 M formic acid was used to maximise protonation of analytes and isasserted to be generally applicable to simultaneous analysis of otherclasses of small, polar molecules with differing pKa values.

The publication of Kellmann and Neilan (2007) discloses the fermentativeproduction of neosaxitoxin and its analogs in recombinant Escherichiacoli strains.

The publications of Lagos Gonzáles (2010, 2015a, 2015b and 2016)disclose the purification of phycotoxins from cyanobacteria produced ina continuous culture. The phycotoxins are isolated primarily from thebacteria, but can also be isolated from the culture medium. In oneembodiment of the process disclosed only neosaxitoxin (neoSTX) andsaxitoxin (STX) are produced. In another embodiment of the processdisclosed only gonyautoxin 2 (GTX2) and gonyautoxin 3 (GTX3) areproduced.

The publication of Wang et al (2010) discloses the preparation of aparalytic shellfish poison (PSP) standard solution. The standardsolution is prepared by removing impurities from shellfish material,collecting shellfish meat, adding distilled water and 0.1-0.3 mol/Lhydrochloric acid solution, regulating pH to 1.5 to 5.0, andhomogenising to obtain homogenate, precooling at −20° C. for 30 minutesto 24 hours, and lyophilising to obtain a core sample, grinding, andsieving, precooling at −20° C. for 10 minutes to six hours andlyophilising to obtain the standard sample. The method of preparation isasserted to have the advantages of low raw material cost and a simplepreparation process.

The publication of Xiong and Qiu (2009) discloses the application ofbiguanido purine derivatives and their salts and esters for improvingthe therapeutic effect and reducing the side effects of antitumoragents. The biguanido purine derivates are saxitoxin analogs.

It is an object of the invention to provide a method of preparingneosaxitoxin in sufficient quantity and of sufficient purity to enableits use in the manufacture of pharmaceutical preparations. This objectis to be read in the alternative with the object to at least provide auseful choice.

SUMMARY OF INVENTION

In a first aspect a method of preparing a volume of concentrated aqueousextract for use in the preparation of a quantity of GTX1,4 is described,the method comprising the steps:

-   1. Culturing a selected isolate of a dinoflagellate in a vertical    column of aerated amended seawater for a period of time and at a    temperature sufficient to provide a culture having a predetermined    cell density;-   2. Harvesting the cells from the culture having the predetermined    cell density to provide a quantity of cellular biomass;-   3. Resuspending the quantity of cellular biomass in an aqueous    solution of a weak organic acid for a period of time and at a    temperature sufficient to provide a mixture of residual biomass and    an extract in solution;-   4. Separating the residual biomass from the extract in solution; and    then-   5. Reducing the volume of the extract in solution to provide the    volume of concentrated aqueous extract,

where the selected isolate has been selected to produce a ratio ofGTX2,3 to GTX1,4 of less than 0.1, the amended seawater is seawateramended with a nutrient medium, and the predetermined cell density is inthe range 7×10⁴ to 10⁵ cells/mL.

In an embodiment the selected isolate is an isolate of thedinoflagellate Alexandrium pacificum. The isolate is may be an isolateof the dinoflagellate Alexandrium pacificum that produces a ratio ofGTX2,3 to GTX1,4 of less than 0.01. The isolate may be the isolate ofthe dinoflagellate Alexandrium pacificum designated CAWD234.

The nutrient medium may comprise nitrates, phosphates, trace metals andvitamins.

The aqueous solution of a weak organic acid may be 0.25 to 0.75% aceticacid.

The volume of concentrated aqueous extract may have a density between1.06 to 1.14 g/mL.

In a second aspect a method of fractionating a volume of concentratedaqueous extract to provide a solution of partially purified GXT1,4 isdescribed, the method comprising the steps:

-   1. Reducing the volume of the aqueous extract by ultrafiltration to    provide a reduced volume;-   2. Loading the reduced volume on a column of activated carbon    sorbent to provide a loaded column; and-   3. Eluting the loaded column with a stepwise gradient of water    followed by aqueous acetic acid/acetonitrile to provide the solution    of partially purified GTX1,4,

where the volume of concentrated aqueous extract is an extract of aculture of a dinoflagellate.

The volume of concentrated aqueous extract may be an extract of aculture of a dinoflagellate prepared according to the method describedas the first aspect.

In a third aspect a method of preparing a quantity of neoSTX isdescribed, the method comprising the step of contacting in solution in areaction solvent a quantity of GTX1,4 and a quantity of dithiol for aperiod of time and at a temperature sufficient to provide a conversionproduct in which greater than 97.5% (w/w) of the GTX1,4 has beenconverted to neoSTX.

The quantity of GTX1,4 is typically of a purity of at least 97.5% (w/w).The quantity of GTX1,4 may be of a purity of at least 98.75% (w/w) or atleast 99% (w/w). The pH of the solution is in the range 7.2 to 7.8 andmay be in the range 7.4 to 7.6. The reaction solvent may be bufferedaqueous acetic acid. The dithiol may be selected from the groupconsisting of dithiothreitol (DTT) and dithiobutylamine (DTBA). In anembodiment the dithiol is dithiothreitol (DTT).

The conversion product may be applied to a silica based weak cationexchange sorbent and eluted with an aqueous weak acid to separate theneoSTX from the dithiol and provide the quantity of neoSTX. The aqueousweak acid may be an aqueous weak organic acid, such as aqueous aceticacid.

The quantity of neoSTX may be greater than 100 mg with a purity greaterthan 99.5% (w/w). The method of preparing the quantity of neoSTX may bea near quantitative method. The method provides for the batchpreparation of neoSTX in a quantity and of a purity not previouslyobtainable (cf. Lagos Gonzáles (2010, 2015a, 2015b and 2016)).

In the description and claims of this specification the followingabbreviations, acronyms, phrases and terms have the meaning provided:“batch preparation” means prepared discontinuously; “biosynthetic” meansprepared within living organisms or cells; “CAS RN” means ChemicalAbstracts Service (CAS, Columbus, Ohio) Registry Number; “comprising”means “including”, “containing” or “characterized by” and does notexclude any additional element, ingredient or step; “consisting of”means excluding any element, ingredient or step not specified except forimpurities and other incidentals; “consisting essentially of” meansexcluding any element, ingredient or step that is a material limitation;“GTX” means gonyautoxin; “GTX1” mean gonyautoxin 1 [CAS RN 60748-39-2];“GTX4” means gonyautoxin 4 [CAS RN 64296-26-0]; “GTX1,4” means anunresolved mixture (as solid or in solution) comprising gonyautoxin 1and gonyautoxin 4; “GTX2,3” means an unresolved mixture (as solid or insolution) comprising gonyautoxin 2 and gonyautoxin 3; “nearquantitative” means greater than 97.5% (w/w) of substrate, e.g. GTX1,4,is converted to product, e.g. neoSTX; “neoSTX” means(3aS,4R,10aS)-2-amino-4-[[(aminocarbonyl)oxy]methyl]-3a,4,5,6,8,9-hexahydro-5-hydroxy-6-imino-1H,10H-pyrrolo[1,2-c]purine-10,10-diol[CAS RN 64296-20-4]; “nutrient medium” means a medium comprising tracemetals and vitamins; “preparative scale” means prepared in batches ofgreater than 100 mg; and “semi-synthetic” means prepared by chemicalconversion of an at least partially purified biosynthetic precursor. Aparonym of any of the defined terms has a corresponding meaning.

The terms “first”, “second”, “third”, etc. used with reference toaspects, elements, features or integers of the matter described in theSummary of Invention, or with reference to alternative embodiments, arenot intended to imply an order of preference. Where concentrations orratios of reagents are specified the concentration or ratio specified isthe initial concentration or ratio of the reagents. Where a pH or pHrange is specified, the pH or pH range specified is the initial pH or pHrange. Where values are expressed to one or more decimal places standardrounding applies. For example, 1.7 encompasses the range 1.650 recurringto 1.749 recurring. Purity of the quantity of neoSTX is determinedaccording to Method 3 [F. Analysis].

The methods will now be described in detail with reference toembodiments or examples and the figures of the accompanying drawingspages. Although the description is provided referencing specificembodiments or examples it should be appreciated that variations andmodifications may be made to these specific embodiments or examples.Where known equivalents exist to specified elements, features orintegers of the embodiments or examples, such equivalents areincorporated as if specifically referred to in the description.Variations and modifications to the embodiments or examples that includesubstitution of elements, features or integers described for equivalentelements, features or integers disclosed in and selected from thereferenced publications are within the scope of the protection soughtunless specifically excluded. The advantages provided by the methodsdescribed and discussed in the description may be provided in thealternative or in combination in different embodiments.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. A plan view of a hanging bag (1) formed from a length of tubularplastic for use in the bulk culture of isolates. Traversing single (3,4, 5 and 6) and double solid lines (9 and 10) identify where two or morelayers of the tubular plastic are heat welded together. Traversingbroken lines identify where four (7 and 8) or two (12 and 13) layers ofthe tubular plastic are cut to provide a hanging loop (2) and cone,respectively.

FIG. 2. The profiles of toxins produced by individual isolates (Table 1)when cultured in vertical columns of aerated amended sea water. Theisolates designated as CAWD12, CAWD20 and CAWD121 are identified asnon-producers of toxins under these culture conditions.

FIG. 3. Amounts of toxins produced by individual isolates (Table 1) whencultured in vertical columns of aerated amended sea water.

FIG. 4. Plot of concentration of gonyautoxins (GTX 1 (♦) and GTX 4 (▪))versus time.

DESCRIPTION

According to the methods described a quantity of GTX1,4 is purified on apreparative scale, i.e. in batch quantities of greater than 100 mg, froma concentrated extract of a culture of a dinoflagellate and converted ina solution buffered to a pH around 7.5 to neoSTX by reductivedesulfonation using a dithiol as the preferred reducing agent.

Extracts of cultures of isolates of Alexandrium pacificum that producerelatively low amounts of gonyautoxin 2 (GTX2) and gonyautoxin 3 (GTX3)have been determined to be most suitable as a source for thepurification of GTX1,4 on a preparative scale. Extracts of cultures ofdinoflagellates that may be used for the purification of GTX1,4 inaccordance with the method described here are available from theCawthron Institute, Nelson, New Zealand.

The introduction of an ultrafiltration step prior to desalting has beenfound to be particularly advantageous when purifying GTX1,4 from theseaqueous extracts on a preparative scale. Without wishing to be bound bytheory it is believed that ultrafiltration removes a substantial portionof the solutes that might otherwise interfere with the desalting step onsorbents such as activated carbon. The introduction of theultrafiltration step thereby reduces the quantity of sorbent that wouldotherwise be required.

The publication of Laycock et al (1994) discloses the extraction andpurification of GTX1 and GTX4 from hepatopancreas of scallops(Placopecten magallanicus):

-   -   Tissues (1 kg) were homogenized in 1 L of 0.1 M HCl using a        Polytron tissue homogenizer, (Model PT10/35, Brinkman        Instruments Canada Ltd, Rexdale, ON). The slurry was heated to        80° C. for 30 min, then cooled and centrifuged (5,000 g, 20 min)        to remove precipitated protein. The supernatant fluid was        extracted twice with dichloromethane (500 ml each). The aqueous        layer was concentrated by rotary evaporation to 200 ml then        poured onto a column (10 cm ID×15 cm) of a mixture of activated        charcoal (Norite, A, 500 g, BDH Ltd.) and Celite (500 g,        Johns-Manville). The column was washed with a solution of 20%        ethanol and 1% acetic acid. Several one liter fractions were        collected and toxin concentrations monitored by HPLC-FD.        Toxin-containing fractions were concentrated by rotary        evaporation and lyophilized.

The publication further discloses separation on Bio-Rex-70 was notcomplete for any of the gonyautoxins. However, by repeatedly removingGTX2 and re-equilibrating the mixture, the proportion of GTX2 and GTX3contaminating the GTX1 and GTX4 fractions was gradually reduced.

The publication of Laycock et al (1995) discloses that dithiothreitol ata concentration of 100 mM in aqueous solution at pH 8.5 rapidlyconverted GTX1,4 to neoSTX and a small (less than 10%) amount ofneosaxitoxinol (as determined by capillary electrophoresis). Bycontrast, it has now been determined that when performing the conversionat a preparative—as opposed to analytical—scale the optimum pH is lowerand in the range 7.2 to 7.8, more specifically 7.4 to 7.6, whendithiothreitol (DTT) is used as the reducing agent.

In solution, gonyautoxin 1 (GTX1) and gonyautoxin 4 (GTX4) exist as apair of epimers of which GTX1 is the thermodynamically most favoured.Epimerisation is believed to occur under most conditions via keto-enolequilibration at C-12. In the first step of this postulated 2-stepreaction mechanism according to SCHEME I a thiol group of the dithiol(R-SH) attacks the electrophilic C-12 of the keto form (I) to form athiohemiketal (II). Conversion to a thioether (IV) occurs via anepisulfonium ion intermediate (III) when the leaving group (O-sulfate)is oriented anti to the sulphur atom (as in the reactive epimer GTX1).In the second step of the proposed reaction mechanism the thiol group ofthe dithiol reacts with the sulphur of the thioether (IV) to form adisulfide thereby yielding an enolate that readily hydrates to neoSTX(V).

The optimal pH for the conversion of GTX1,4 to neoSTX described in thefollowing example has been determined to be around 7.5. Without wishingto be bound by theory it is believed that a pH in this range ensuresboth (i) an optimal rate of epimerisation between the gonyautoxinepimers and (ii) optimal degrees of electrophilicity at C-12 anddeprotonation of the dithiol used as the reducing agent. The use ofdithiols such as dithiothreitol (DTT) and dithiobutylamine (DTBA) ispreferred over the use of monothiols such as glutathione (GSH) andmercaptoethanol (ME) (cf. Sakamoto et al (2000) and Sato et al (2000)).Higher rates of conversion are obtained when using the dithiols,rendering them more suitable for use in the production of neoSTX on apreparative scale.

The excess dithiol, sodium phosphate buffer and unreacted GTX1,4 hasbeen found to be most conveniently removed from the neoSTX containingconversion product by

the use of cation exchange chromatography. The silica based weak cationexchange sorbent Sepra™ WCX has been determined to be a suitable sorbentas it has been determined not to retain DTT. Trials of the polymericbased weak cation exchange sorbent Strata-X™ CW (Phenomenex) determinedthis sorbent to be unsuitable for purification of neoSTX from theconversion product on a preparative scale. The excess dithiol isretained by both an ion exchange and a reverse phase mechanism whenusing this sorbent. Although a portion of the excess DTT is eluted withorganic solvents such as acetonitrile/water a further portion is elutedwith 1 M acetic acid frustrating the purification of the neoSTX whenusing this sorbent.

Example

A. Materials

10 mM acetic acid (0.6 g/L in deionised water); amended seawater (7 mL/LL1 nutrient medium in seawater); deionised water (Milli-Q™,Merck-Millipore); L1 nutrient medium (1.25 g/L EDTA, 0.91 g/LFeCl₃.6H₂O, 0.29 mL/L trace metal stock solution, 21.6 g/L NaNO₃, 1.44g/L NaH₂PO₄ and 14.4 mL/L vitamin stock solution); Mobile Phase A (2.2g/L sodium heptane sulphonic acid (sodium salt) and 0.31 g/L 85%phosphoric acid adjusted to pH 7.1 with 25% ammonium hydroxide); MobilePhase B (0.1% acid in acetonitrile); Mobile Phase C (0.1% acetic acid indeionised water); roll of tubular (230 mm×200 m×250 μm) low densitypoly(ethylene) (LDPE) plastic (Amcor Limited); seawater (30 to 37 pptsalinity); trace metal stock solution (2.5 g/L CuSO₄.5H₂O, 20 g/LNa₂MoO₄.2H₂O, 23 g/L ZnSO₄.7H₂O, 11.9 g/L CoCl₂.6H₂O, 178 g/LMnCl₂.4H₂O, 1.3 g/L H₂SeO₃, 2.6 g/L NiSO₄.6H₂O, 1.8 g/L Na₃VO₄ and 1.9g/L K₂CrO₄); vitamin stock solution (0.01 g/L biotin, 2 g/L thiamine and0.01 g/L vitamin B12).

Vitamin stock solutions are filter sterilised and aseptically addedthrough a 0.22 μm syringe filter during preparation of amended seawaterfollowing the autoclaving of other ingredients.

B. Inocula

Isolates are obtained from naturally occurring algal blooms in coastalwaters. Species responsible for harmful algal blooms include Alexandriumminutum, Alexandrium pacificum (formerly referred to as Alexandriumcatenella), Alexandrium tamarense and Gymnodium catenatum.

The dinoflagellate Alexandrium pacificum is an armoured, marine,planktonic dinoflagellate. The species was originally identified asAlexandrium catenella using the morphological characteristics describedin the publication of Balech

TABLE 1 Isolates evaluated for their production of toxins and isolatedfrom New Zealand coastal waters prior to October 2015. OriginalDesignation identification Provenance CAWD11 Alexandrium minutum AnakoheBay, New Zealand CAWD12 Alexandrium minutum Anakohe Bay, New ZealandCAWD20 Alexandrium tamarense Tamar East, Plymouth, United Kingdom CAWD44Alexandrium catanella Tauranga, New Zealand CAWD45 Alexandrium catanellaTauranga, New Zealand CAWD46 Alexandrium catanella Tauranga, New ZealandCAWD47 Alexandrium catanella Tauranga, New Zealand CAWD49 Alexandriumcatanella Te Kaha, New Zealand CAWD50 Alexandrium catanella Te Kaha, NewZealand CAWD101 Gymnodinium catenatum Kaitaia Spat, New Zealand CAWD102Gymnodinium catenatum Kaitaia Spat, New Zealand CAWD121 Alexandriumtamarense Marsden Point, New Zealand CAWD234 Alexandrium catanella OpuaBay, New Zealand CAWD235 Alexandrium catanella Opua Bay, New Zealand

(1985). A more recent description of the characteristics of species ofthe genus is provided in the publication of MacKenzie et al (2004).

A chain-forming species, Alexandrium pacificum typically occurs incharacteristic short chains of 2, 4 or 8 cells. Single cells are round,slightly wider than long, and are anterio-posteriorly compressed. Asmall to medium sized species, it has a rounded apex and a slightlyconcave antapex. The thecal plates are thin and sparsely porulated.Cells range in size between 20 to 48 μm in length and 18 to 32 μm inwidth.

As stated in the publication of MacKenzie (2014), Alexandrium pacificumis the cause of most paralytic shellfish toxin contamination in NewZealand. Individual isolates (Table 1), including isolates of thespecies Alexandrium pacificum, have been evaluated for their productionof toxins when cultured in bulk according to the following protocols.Representations of the structures of the toxins referred to in FIG. 2and FIG. 3 are provided below. The representations are of the structuresof the toxins in their neutral (uncharged) form (and not necessarily themost thermodynamically favoured tautomeric form).

C. Culture

Individual isolates are cultured in aerated vertical columns of amendedseawater in a plurality of hanging bags. The bags are hung in anincubation room maintained at a temperature of 16 to 20° C. The bags areilluminated according to a 16-hour/8-hour light/dark cycle. Automatedcarbon dioxide (CO₂) dosing is used to maintain the pH in the range 8 to9 during the light phase of the

light/dark cycle. Standard personal protection equipment is worn byoperators to minimise the risk of exposure to toxins.

Reference is made to FIG. 1 of the accompanying drawings pages where aplan view of a cut and welded hanging bag (1) for use in the bulkculture of isolates is provided. The bags are formed from a roll oftubular plastic by cutting and heat welding according the followingprotocol.

After discarding the first two metres of a new roll of tubular plastic a20 cm section is cut and the open ends of the excised section eachsealed by a heat weld. The inner surfaces of the sealed section oftubular plastic roll are subjected to microbiological evaluation beforethe remainder of the new roll of tubular plastic is used for thefabrication of hanging bags.

A two-metre length (A+G) is dispensed from the tubular plastic roll anda hanging loop (2) formed at the first end. The hanging loop (2) isformed by folding back a 10 cm section (A) of the tubular plastic andheat welding together the four layers of plastic to provide a first heatweld (3) proximal to the cut first end. A second heat weld (4) is formedwithin the folded back section (A) parallel and spaced apart from thefirst heat weld (3) by about 2 cm followed by diagonal heat welds (5,6)traversing the region between the horizontal first and second heat welds(3,4). The corners of the folded back section distal from the welds arethen cut (7,8) to provide a hanging loop (2) of a width (B) capable ofsupporting the length of tubular plastic when filled with a volume ofamended seawater.

The second end of the length of tubular plastic is sealed by doublediagonal heat welds (9,10) converging to a point (11) proximal to thecentre of the second end. The integrity of each double diagonal heatweld (9,10) is inspected visually before each triangle of plasticoutside the conical sealed end is cut (12,13) away. The conical portionof the sealed end has a depth (C) of around 20 cm. Each hanging bag (1)is capable of containing a culture volume of approximately 24 L

Prior to filling with inoculum and amended seawater a hanging bag (1) ishung in position and the top outside corner surface sterilised by wipingwith isopropanol. A downward pointing first hole is formed in thesurface sterilised region using a sterile pick. The tip of a sterilisedair vent inserted into the hole and taped to the outside of the bagusing PVC insulation tape (50 mm width). A region of the outer surfaceof the conical sealed end is also surface sterilised by wiping withisopropanol. A second hole is formed in the surface sterilised regionusing a sterile pick. The downward pointing tip of a sterile inoculationline is inserted into the hole and the line taped to the outside of thebag using PVC insulation tape (50 mm width).

Inoculum is fed into the hanging bag from a parent culture via theinoculation line. The line from the parent culture and the inoculationline to the hanging bag are each connected to a manifold. Air is purgedfrom the lines by pumping amended seawater into both. The parent cultureis then allowed to flow into the hanging bag. Pressurised air isintroduced into the hanging bag containing the parent culture via itssterilised air vent. Equal volumes of the parent culture are transferredto multiple hanging bags. The inoculum containing hanging bags are thenfilled with the amended seawater to a fill line (D). Once the hangingbags are filled, the inoculation lines are disconnected from themanifold and connected to an air line via a sterilised air filter. Theculture volume is aerated via the air line.

To monitor the pH of the medium during the culture the surface of a pHprobe, including the glass bulb, is washed with deionised water and 70%(w/w) ethanol. A region of the outer surface of the hanging bag around 4cm above the fill line is surface sterilised by wiping with isopropanoland a hole made in the bag within this region using a sterilised 10 mLpipette tip. The pH probe is inserted via the hole and into the culturevolume and held flat against the inner side wall of the bag.

Under these incubation conditions the isolates have a doubling time ofabout two days and are harvested when the cell density has reached 7×10⁴to 10⁵ cells/mL as determined by microscopic image cytometry.

D. Harvest

With aeration maintained the pH probe is removed and a volume of about 9mL glacial acetic acid introduced into the culture volume. After about10 to 20 minutes a volume of about 100 mL of a suspension of hydratedbentonite clay is added to the culture to provide a dosage of about 4mL/L of culture. After a further 5 to 10 minutes the inoculation line isclamped and disconnected from the air filter. Settling of cells occursover a period of time of at least 2 hours. The settled cells are drainedinto a centrifuge bottle and the collected volume (300 to 400 mL/bag)centrifuged in balanced bottles at a force of 1,500×g for a period oftime of 5 minutes. The supernatant is discarded, and the harvested cellsweighed before storing frozen at −20° C.

E. Extraction

Extracts for (i) determining toxin profiles and monitoring toxinproduction, or (ii) preparation of GTX1,4 are prepared according to thefollowing methods.

For monitoring toxin production, 10 mL of the culture volume istransferred to a polypropylene centrifuge tube and the cells pelleted bycentrifugation at a force of 1,500×g for a period of time of 5 minutes.The supernatant is discarded, and the pellet resuspended in a volume of250 μL 1 mM acetic acid. The volume is sonicated for a period of time of2 minutes and heated to 80° C. for a period of time of 10 minutes. Thecellular material is pelleted by centrifugation at a force of 3,220×gfor a period of time of 5 minutes and 10-fold and 20-fold dilutions ofthe supernatant then prepared in 80% (v/v) acetonitrile/0.25% (v/v)acetic acid.

For preparation of GTX1,4, the frozen pellet of harvested cells isdefrosted before resuspending in an equal volume of 0.5% acetic acid andleaving at room temperature for a period of time of 30 to 60 minutes.The suspension in acetic acid is then heated and maintained at atemperature of 85±2° C. in a water bath for a period of time of 10 to 15minutes. The heated suspension is then cooled in an ice slurry beforecentrifugation at a force of 3,990×g for a period of time of 2 minutesand the first supernatant decanted to a collection vessel. An equalweight of 0.5% acetic acid is added to the pelleted cellular material,mixed well, and centrifuged at a force of 3,990×g for a period of timeof 5 minutes. The second supernatant is decanted to the collectionvessel and the total volume of collected supernatants reduced by rotaryevaporation under vacuum (less than 15 mBar) at a temperature of 30° C.(For example, portions of a total volume of 10 litres of supernatant aretransferred from the collection vessel to a weighed 5 litre round bottomflask and the volume reduced by rotary evaporation until an extracthaving a weight of 800±50 g and a density between 1.08 to 1.12 g/mL isobtained.) The concentrated extract is stored frozen at −20° C.

F. Analysis

A sample of extract is prepared for analysis using activated carbonsolid phase extraction (SPE). A Supelclean™ ENVI-Carb™ SPE tube (bed wt.250 mg, volume 6 mL) is conditioned with a volume of 3 mL of 20%acetonitrile/1% acetic acid followed by a volume of 3 mL of 0.025%ammonia. Following elution to the top frit the tube is loaded with atotal volume of 400 μL consisting of 10 to 400 μL of extract and quantumsufficit deionised water. The cartridge is eluted to the top frit usinga vacuum of −15 to −20 kPa and washed with a volume of 3 mL of deionisedwater before elution with a volume of 5 mL of 20% acetonitrile/1% aceticacid. The eluate is collected in a polypropylene tube and a volume of 10μL diluted 4-fold with the addition of acetonitrile in a polypropyleneautosampler vial. The contents of the vial are mixed on a vortex mixturewith further dilution as required.

Method 1

Aqueous extracts and fractions enriched for the presence of GTX1,4 areanalysed by high pressure liquid chromatography (HPLC) (ShimadzuProminence) with ultraviolet (UV) detection according to this Method 1.

Samples are diluted with 10 mM acetic acid to provide a nominalconcentration of 200 μg/mL. A volume of 40 μL of diluted sample isinjected onto a column (4.6×150 mm) of 3.5 μm Zorbax Bonus RP elutedwith Mobile Phase A at a flow rate of 1 mL/min for a period of time of30 minutes while being maintained at a temperature of 20° C. Theabsorbance of the eluate is monitored at wavelengths of 210 nm (purity)and 245 nm (quantity) using a photodiode array detector (PAD). Thepurity of samples is calculated based on the percentage area at 210 nmand retention time with reference to a standard at a concentration ofapproximately 200 μg/mL GTX1,4. Samples are analysed in triplicate with10 mM acetic acid used as a blank and the chromatogram obtainedsubtracted from that obtained for all samples.

Method 2

Extracts and fractions enriched for the presence of GTX1,4 are analysedand quantified by liquid chromatography-mass spectrometry(LC-MS)(Shimadzu 8050) according to this Method 2.

Samples are diluted to an appropriate concentration using 80%acetonitrile/0.25% acetic acid. A mixed standard containing a number ofreference paralytic shellfish toxins (PSTs) is used. A maximum volume of2 μL of diluted sample is injected by means of an auto samplermaintained at 4° C. onto a column (2.1×100 mm) of 1.7 μM Waters AcquityUPLC BEH amide eluted at a flow rate of 0.4 mL/min while beingmaintained at a temperature of 20° C. The column is eluted stepwise with75% Mobile Phase B/25% Mobile Phase C for a period of time of 5 minutesfollowing injection, followed by 55% Mobile Phase B/45% Mobile Phase Cfor a period of time of 0.50 min before reverting to 75% Mobile PhaseB/25% Mobile Phase C. The eluate is monitored by mass spectrometry usingselective ion monitoring in ESI− and ESI+ ionisation modes.

Method 3

Purified products of extraction, fractionation and conversion areanalysed for quantity and purity according to this Method 3.

Isolated products (GTX1,4 or neoSTX) are diluted to a concentration of200 μg/mL in 10 mM acetic acid. The diluted sample is then furtherdiluted 100-fold in 8% acetonitrile/0.25% acetic acid to provide asolution of product at a concentration of 20 mg/mL for quantitativeanalysis. A mixed standard containing a number of reference paralyticshellfish toxins (PSTs) is also prepared in the same solvent. A solutionof 2 μL of the diluted product (20 ng/mL) is injected by means of anautosampler maintained at a temperature of 40° C. onto a column (2.1×100mm) of 1.7 μm Waters Acquity UPLC BEH amide eluted at a flow rate of 0.6mL/min while being maintained at a temperature of 60° C. The column iseluted stepwise with 80% Mobile Phase B/20% Mobile Phase C for a periodof time of 6 minutes following injection, followed by 55% Mobile PhaseB/45% Mobile Phase C for a period of time of 0.50 minutes beforereverting to 80% Mobile Phase B/20% Mobile Phase C. The eluate ismonitored by mass spectrometry monitoring in ESI− and ESI+ ionisationmodes.

G. Purification

The concentrated extract is thawed at room temperature. The extract isdivided between two balanced 500 mL conical centrifuge bottles andcentrifuged at a force of 4000×g for a period of time of 10 minutes. Thesupernatant is decanted and filtered under reduced pressure through aseries of 110 mm diameter hardened ashless and glass microfiber filterpapers (Whatman™ grade 540, Whatman™ grade 542 and Whatman™ grade GF/A).

The filtered extract is then subjected to crossflow ultrafiltration byrecirculation through two filters (VivaFlow 200) connected in parallelwith an outlet pressure no greater than 2.5 bar until the volume of theextract has been reduced to a volume of 10 to 20 mL of retentate.

The retentate is transferred to a 100 mL bottle and made up to a totalvolume of 100 mL with deionised water. The diluted retentate issimilarly subjected to rounds of crossflow ultrafiltration reducing thevolume of retentate to 10 to 20 mL before making up to a total volume of100 mL with deionised water.

A 32 mm 5 μm syringe filter (Pall Corp.) is installed on the inlet of a50 g Sepabeads SP-270 (Supelco) SPE cartridge conditioned with 500 mL ofdeionised water at a flow rate of 30 mL/minute. The washed retentate ispassed through the conditioned SPE cartridge at a flow rate of 30mL/minute and the effluent collected. The SPE cartridge is then elutedwith 200 mL of deionised water at a flowrate of 30 mL/minute and theeffluent collected.

The volume of the combined effluents is reduced by rotary evaporationunder vacuum (less than 15 mBar) at a temperature of 30±2° C. The volumeis reduced to provide a weight of 500±50 g, the density determinedgravimetrically, and the total reduced volume calculated on this basis.

Prior to purification of the GTX1,4 from the reduced volume on apreparative scale a sample of the reduced volume is prepared andanalysed as described (F. Analysis). A 10,000-fold dilution of thesample should provide a concentration of GTX1,4 in the range 20 to 50ng/mL. The calculated yield of the GTX1,4 should exceed 90% (w/w).

The total reduced volume is desalted by loading on a 100 g 25×450 mmcarbon column (Enviro-Clean™ Graphitized Carbon Non-Porous, UCT)conditioned with 1 L of deionised water. The reduced volume is loaded ata flow rate of 30 mL/min using a Mini-Flash Pump (Sorbent Technologies,Inc.) and eluted at a flow rate of 15 mL/min with a stepwise gradient ofdeionised water for a period of time of 40 minutes followed by 0.2%(v/v) acetic acid/30% (v/v) acetonitrile for a period of time of 40minutes.

The eluate is monitored at 205 nm using an inline UV detector andsequential volumes of 10 mL of eluate collected as fractions. Volumes of5 μL of selected fractions are diluted to a total volume of 10 mL with0.25% (v/v) acetic acid/80% (v/v) acetonitrile and submitted to LC-MSanalysis as described (F. Analysis). The GTX1,4 containing fractions arecombined.

The pH of the desalted combined fractions collected from the carboncolumn is adjusted to 7.8 using concentrated ammonium hydroxide at arate of approximately 2 μL/mL. The pH adjusted volume is then loaded ona 35×460 mm column of 280 g Sepra™ WCX-NH₄ ⁺ conditioned using a volumeof 1 L of 50 mM ammonium bicarbonate at a flow rate of 30 mL/min. Thecolumn is eluted with a gradient of 10 to 80% 0.5 M ammonium bicarbonateover a period of time of 100 minutes. The eluate is monitored atwavelengths of 205 nm and 245 nm using an inline UV detector. Sequentialvolumes of 25 ml are collected as fractions and combined according tothe UV monitoring and LC-MS analysis where required (F. Analysis).Fractions containing greater than 2% of the total amount of GTX1,4 arecombined and the pH of the combined fractions reduced to 6.5 by thedropwise addition of glacial acetic acid. A 5,000-fold dilution of theacidified volume is subjected to LC-MS analysis as described (F.Analysis) and the total quantity and yield of GTX1,4 and the ratio toGTX5 calculated. The yield should be greater than 90% and the ratio ofGTX5 to GTX 1,4 should be less than 1%.

The combined fractions from the weak cation exchange chromatography areloaded onto a 35×480 mm column of 175 g Sepra™ ZT-WCX-H⁺ formconditioned using 1 L of deionised water at a flowrate of 30 mL/min. Theloaded column is eluted with a continuous gradient of 0 to 100% 1Macetic acid in water over a period of time of 100 minutes and sequentialvolumes of 25 mL of eluate collected as fractions while monitoring at awavelength of 205 nm using an inline UV detector.

The GTX1,4 containing fractions are collected from baseline to baselineand the total volume reduced to a volume of 10 to 20 mL by rotaryevaporation at 30° C. under reduced pressure of less than 15 mBar. A500,000-fold dilution of the reduced volume is subjected to LC-MSanalysis as described (F. Analysis) and the total quantity and yield ofGTX1,4 calculated on this basis.

The GTX1,4 containing reduced volume is loaded on a 50×500 mm column ofP2 gel conditioned with 2 L of 100 mM acetic acid at a flowrate of 50mL/min and eluted isocratically with the same conditioning mobile phase.The eluate is monitored at a wavelength of 205 nm for a period of timeof 200 minutes using an inline UV detector and sequential volumes of 10mL of eluate collected as fractions. The GTX1,4 containing fractions arecollected from baseline to baseline and the total volume reduced byrotary evaporation at a temperature of 30° C. under reduced pressure ofless than 15 mBar.

The reduced volume of combined fractions obtained by gel filtration istransferred to a pre-weighed 100 mL round bottom flask and evaporated todryness by rotary evaporation at a temperature of 30° C. under a reducedpressure of less than 15 mbar followed by drying in a freeze dryer at ashelf temperature of 10° C. and pressure of 0.05 mbar for 24 hours. Theopen mouth of the round bottom flask is securely covered with an airpermeable, lint free tissue before placing in the freeze dryer. Theyield of purified GTX1,4 is determined gravimetrically. The purifiedGTX1,4 is dissolved in a known volume of deionised water to provide asolution containing 70 to 100 mg/mL of GTX 1,4.

A 500-fold dilution of the solution in 10 mM acetic acid is prepared andanalysed as described (F. Analysis). The final volume required toprovide a concentration of 40 to 45 mg/mL of GTX 1,4 is calculated andthe solution transferred via a filter to a pre-weighed 10 mL amber glassvial, rinsing with deionised water to provide a transferred volumehaving the target concentration of 40 to 45 mg/mL. A 200-fold dilutionof the transferred solution is prepared in 10 mM acetic acid andanalysed as described (F. Analysis). The dilution is diluted a further10-fold in 10 mM acetic acid and analysed for purity as described (F.Analysis).

H. Conversion

A quantity of 183 mg (as the free base) of GTX1,4 purified according tothe preceding steps from an extract of a culture of the isolate ofAlexandrium pacificum designated CAWD234 is dissolved in a total volumeof 5 mL of dilute acetic acid and mixed with a volume of 45 mL of 0.2 Mphosphate buffer at a pH of 7.5 in a 100 mL round bottom flask. Themixture is placed on ice and the pH adjusted from 6.8 to 7.5 with theaddition whilst stirring of solid sodium carbonate. A quantity of 1.5 gof dithiothreitol (DTT) is added to the pH adjusted mixture and itsdissolution promoted by placing the reaction mixture containing roundbottom flask in an ultrasonic bath before transferring to a water bathmaintained at a temperature of 50° C. Aliquots of a volume of 10 μL areremoved from the reaction mixture and transferred to the water bath(T=0) and periodically (every 15 minutes) thereafter. Aliquots arediluted 50-fold by the addition of a volume of 490 μL 80% acetonitrile0.25% acetic acid immediately following removal from the reactionmixture and analysed by LC-MS as described (F. Analysis) to monitor theprogress of the reaction in near real time (FIG. 4). After incubationfor 45 minutes at 50° C. the reaction mixture is chilled by transferringthe round bottom flask to an ice slurry. Under these conditions nearquantitative conversion of GTX1,4 to neoSTX is observed with close to100% yield.

I. Isolation

The conversion product is loaded onto a quantity of 39 g Sepra™ WCXpacked in an empty flash cartridge (Grace) and preconditioned with avolume of 250 mL of 50% (w/w) acetonitrile followed by a volume of 250mL of deionised water. The conversion product is loaded onto the packedcartridge with rinses of deionised water with collection of the effluent(about 200 mL). The dissolution of any crystals formed during storage ofthe conversion product at 4° C. is achieved by the addition of a minimalamount of deionised water. The loaded packed cartridge is then eluted ata rate of 50 mL/min with a total volume of 1.5 L, followed by elutionwith a continuous gradient to 1 M acetic acid over 20 minutes and thecollection of sequential volumes of 10 mL of eluate as fractions whilemonitoring UV absorbance at 205 nm and 254 nm.

TABLE 2 Specification for a batch (CNC00063) of neoSTX preparedaccording to the semisynthetic method described. Component Quantity (mg)% (w/w) neoSTX 118 99.58 L-arginine 0.444 0.37 STX 0.0546 0.05 DTT<0.005 <0.004 TOTAL 119 100

A volume of 5 μL of fractions demonstrating UV absorbance at 205 nm isdiluted 100,000-fold in 80% acetonitrile 0.25% acetic acid and analysedby LC-MS. Fractions confirmed to comprise neoSTX are combined, frozen at−70° C. and lyophilised. The dried neoSTX is dissolved in a small volumeof 10 mM and transferred to a pre-weighed 10 mL glass vial and a volumeof 10 μL analysed. The purity and quantity of a batch (CNC00063) ofneoSTX prepared according to the foregoing method is provided in Table2.

REFERENCED PUBLICATIONS

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1) A method of preparing a quantity of neoSTX comprising: contacting insolution in a buffered reaction solvent a quantity of GTX1,4 and aquantity of dithiothreitol at a temperature and for a time sufficient toprovide a conversion product in which greater than 97.5% (w/w) of thequantity of GTX1,4 has been converted to neoSTX, wherein the pH of thesolution is in the range 7.2 to 7.8. 2) The method of claim 1, furthercomprising: applying the conversion product so provided to a silicabased weak cation exchange sorbent and eluting with an aqueous weak acidto separate neoSTX from dithiothreitol. 3) The method of claim 2,wherein the quantity of neoSTX has a purity greater than 99.5% (w/w). 4)The method of claim 3, wherein the pH of the solution is in the range7.4 to 7.6. 5) The method of claim 4, wherein the reaction solvent isaqueous acetic acid. 6) The method of claim 5, wherein the aqueous weakacid is an aqueous organic acid. 7) The method of claim 6, wherein theorganic acid is acetic acid. 8) The method of claim 7, wherein thequantity of neoSTX is greater than 100 mg.